CLICK ON weeks 0 - 40 and follow along every 2 weeks of fetal development
Fertilization triggers thousands of changes
For more than half a century, studies on the African clawed frog (Xenopus laevis) have helped scientists come to understand the biological underpinnings of life. From embryo development and neurobiology to genetics and disease, the frog has numerous claims to fame. One is the Nobel Prize-winning discovery that adult cells can be reprogrammed. It also once served as the world's only reliable pregnancy test.
For decades Xenopus eggs have been used to investigate molecular events occurring during fertilization. Though much is known from these years of study, scientists still have an incomplete understanding of many of its aspects due to technical limitations — in particular, the lack of a complete picture of all the proteins involved, the function of each, and what happens to each over time.
Now, new technologies are allowing scientists to learn even more about the fundamental processes driving the biology of these homely organisms. Reporting in the Proceedings of the National Academy of Sciences (PNAS), a team led by Harvard Medical School systems biologist Marc Kirschner, describes a new approach for identifying and measuring changes in thousands of Xenopus egg proteins released after fertilization. The moment of fertilization appears to trigger the destruction of a small number of proteins that apply "brakes" on the egg's continued development. Without these "brakes", vast amounts of other proteins are released to prevent other sperm from contnuing to penetrate and attempt fertilization.
Researchers have now made a comprehensive analysis of protein dynamics within an egg cell during the moment of fertilization. Their results can inform studies of molecular behaviors in a wide range of biological systems and help define cellular changes that drive disease.
"We have developed a method that provides us with a critically important ability to quantify and measure absolute levels of proteins and protein modifications in a dynamic, complex system. The method should be widely used in many biological and biomedical studies."
Although much is known from years of study, scientists still have an incomplete understanding of many aspects of fertilization due to technical limitations. Along with colleagues and graduate students in systems biology at Harvard Medical School, Kirschner used a technique to label proteins in Xenopus eggs with barcode-like chemical tags, allowing scientists to analyze thousands of proteins at once using mass spectrometry. When coupled with a new analytical methodology, this approach allowed the team to measure the absolute levels of proteins in a cell and reveal details about a protein's phosphorylation, one of the primary chemical modifications cells use to regulate the activity of a protein.
Taking measurements during and up to 20 minutes after fertilization, researchers found that levels of a small number of low-abundance proteins quickly drop. Within a few minutes, the destruction of these proteins causes the reverse of phosphorylation of a much broader range of proteins throughout the cell — a process that promotes the completion of the cell cycle, including the separation of chromosome copies, which prepares an egg for further growth.
While only around 0.01 percent of the total protein mass of the cell was degraded, the team found that fertilization also triggers the expulsion of 50 times this amount of protein from the cell. Primarily stored in cell compartments near the membrane, these sets of proteins are likely secreted to help prevent fertilization by multiple sperm cells researchers theorize.
This release coincides with a substantial uptick in phosphorylation for numerous signaling proteins and others that play a role in generating contraction waves on the surface of the egg immediately after fertilization. The egg also secretes several protein-degrading enzymes outside the cell. Researchers suspect this helps block multiple fertilization events by destroying sperm-binding proteins. In a somewhat paradoxical finding, the team also observed an increase in proteins that inhibit the activity of protein-degrading enzymes. The reasons for both processes remain unclear and present an avenue for future study.
"We were able to observe both new and previously known features of the cell cycle, but we were also able to untangle other major events happening in parallel," Presler explains. "Fertilization occurs through the coordination of thousands of molecules at once, and for the first time we have an opportunity to understand it." The new methodology enables measurement of the absolute levels of proteins and their phosphorylation using a combination of technology as well as mathematical approaches — a significant improvement over common large-scale protein analysis.
Having significantly improved the detail and scale of protein composition and modification across narrow windows of time — these techniques may be applicable to many biological systems.
Pressler: "To understand and cure disease, we need a more precise understanding of what is happening in normal, healthy processes... this approach is immediately applicable for studying other important questions, such as the differences between cells that are in healthy versus diseased states. Protein biochemistry drives much of the function of a cell, and this methodology can give us a more complete picture of how cells do what they do."
Protein phosphorylation and degradation drive critical events in early embryogenesis and the cell cycle; however, comprehensive and accurate analysis of these changes is currently difficult. Using a mass-spectrometric approach, we present a quantitative view of the protein and posttranslational economy of the fertilization response in the frog egg. Protein degradation affects a small but very important class of proteins, while regulatory phosphorylation and protein release occur on a far larger scale. We have developed broadly applicable analytical methods for phosphorylation that provide absolute quantification with confidence intervals for improved interpretability of posttranslational modification analysis.
Fertilization releases the meiotic arrest and initiates the events that prepare the egg for the ensuing developmental program. Protein degradation and phosphorylation are known to regulate protein activity during this process. However, the full extent of protein loss and phosphoregulation is still unknown. We examined absolute protein and phosphosite dynamics of the fertilization response by mass spectrometry-based proteomics in electroactivated eggs. To do this, we developed an approach for calculating the stoichiometry of phosphosites from multiplexed proteomics that is compatible with dynamic, stable, and multisite phosphorylation. Overall, the data suggest that degradation is limited to a few low-abundance proteins. However, this degradation promotes extensive dephosphorylation that occurs over a wide range of abundances during meiotic exit. We also show that eggs release a large amount of protein into the medium just after fertilization, most likely related to the blocks to polyspermy. Concomitantly, there is a substantial increase in phosphorylation likely tied to calcium-activated kinases. We identify putative degradation targets and components of the slow block to polyspermy. The analytical approaches demonstrated here are broadly applicable to studies of dynamic biological systems.
Authors: Marc Presler, Elizabeth Van Itallie, Allon M. Klein, Ryan Kunz, Margaret L. Coughlin, Leonid Peshkin, Steven P. Gygi, Martin Wühr, and Marc W. Kirschner
The authors declare no conflict of interest.
This work was supported by the National Institutes of Health (HD091846, HD073104, GM103785, GM39565), a Charles A. King Trust Postdoctoral Fellowship, Princeton University and a Burroughs Wellcome Fund and Mallinckrodt Award.
Harvard Medical School has more than 11,000 faculty working in 10 academic departments located at the School's Boston campus or in hospital-based clinical departments at 15 Harvard-affiliated teaching hospitals and research institutes: Beth Israel Deaconess Medical Center, Boston Children's Hospital, Brigham and Women's Hospital, Cambridge Health Alliance, Dana-Farber Cancer Institute, Harvard Pilgrim Health Care Institute, Hebrew SeniorLife, Joslin Diabetes Center, Judge Baker Children's Center, Massachusetts Eye and Ear/Schepens Eye Research Institute, Massachusetts General Hospital, McLean Hospital, Mount Auburn Hospital, Spaulding Rehabilitation Network and VA Boston Healthcare System.
Return to top of page
Xenopus laevis, from Chimanimani, Manicaland, Zimbabwe.
Image credit: Wikipedia.